Quantitative Analysis of Signal Transduction with In-Cell Western Immunofluorescence Assays.

Protein levels and signaling events can be efficiently quantified in many samples with the In-Cell Western (ICW) cell-based assay. This quantitative immunofluorescence method streamlines experimental procedures and data analysis, so hundreds of samples can be processed in parallel with quantitative data output. Cells are cultured in microplates and treated with various drugs or conditions. After fixation and permeabilization of cells in the microplate wells, immunostaining is used to detect target proteins. Secondary antibodies conjugated with IRDye near-infrared (NIR) fluorescent dyes are used for multiplex detection and normalization; cell stains and DNA stains can also be used for cell number normalization. Fluorescent signals reflect the protein expression levels or signaling status of the cell population in each well. ICW assays are a powerful alternative to western blotting. Time-consuming, error-prone steps such as cell lysis, gel electrophoresis, and membrane transfer are eliminated. In situ detection of protein targets in fixed cells provides a relevant cellular context, and enables very rapid and precise quenching of cellular treatments. Results are consistent with western blotting, but precision and reproducibility are enhanced. ICW functional assays are used to analyze protein phosphorylation, monitor the timing and kinetics of signal transduction events, monitor cellular response to agonists and antagonists, and determine IC50 and EC50 values. Modified On-Cell Western (OCW) assays are used for analysis of cell surface proteins, receptor internalization and recycling, and fluorescent ligand binding studies. Here, we describe the basic methodology for In-Cell Western quantitative immunofluorescence assays.